Abstract:

The purpose of the invention is to provide an activated protein
phosphatase 2A (PP2A) in large quantities with high purity by a genetic
engineering and to provide a method for producing a heterodimer
derivative of PP2A which comprises infecting insect cultured cells with a
baculovirus in which a cDNA encoding the catalytic subunit of PP2A
carrying a first tag is integrated together with another baculovirus in
which a cDNA encoding the A subunit of PP2A carrying a second tag is
integrated, incubating the infected cells, disrupting the incubated cells
to obtain a disrupted cell suspension, and then purifying the disrupted
cell suspension with a solid phase carrying a substance capable of
binding to the first tag and another solid phase carrying a substance
capable of binding to the second tag, characterized in that the insect
cells infected with the baculovirus are incubated at a temperature of
from 18 to 22° C.

Claims:

1. A method for producing a heterodimer derivative of protein phosphatase
2A (PP2A), comprising the steps of:infecting insect cultured cells with a
baculovirus in which a cDNA encoding the catalytic subunit of PP2A
carrying a first tag is integrated together with another baculovirus in
which a cDNA encoding the A subunit of PP2A carrying a second tag is
integrated;incubating the infected cells;disrupting the incubated cells
to obtain a disrupted cell suspension; and thenpurifying the disrupted
cell suspension with a solid phase carrying a substance capable of
binding to the first tag and another solid phase carrying a substance
capable of binding to the second tag,wherein the insect cells infected
with the baculovirus are incubated at a temperature of from 16 to
22.degree. C.

2. A method for producing a heterodimer derivative of PP2A as claimed in
claim 1, wherein the catalytic subunit of PP2A is a catalytic
α-subunit or catalytic β-subunit.

3. A method for producing a heterodimer derivative of PP2A as claimed in
claim 1 or 2, wherein the first tag is a polyhistidine tag, and the
second tag is a FLAG tag.

4. A method for producing a heterodimer derivative of PP2A as claimed in
any of claims 1 to 3, wherein the substance capable of binding to the
first tag is Ni, and the substance capable of binding to the second tag
is an anti-FLAG M2 monoclonal antibody.

5. A method for producing a catalytic subunit derivative of PP2A,
comprising the steps of:infecting insect cultured cells with a
baculovirus in which a cDNA encoding the catalytic subunit of PP2A
carrying a tag is integrated;incubating the infected cells;disrupting the
incubated cells to obtain a disrupted cell suspension; and thenpurifying
the disrupted cell suspension with a solid phase carrying a substance
capable of binding to the tag,wherein the insect cells infected with the
baculovirus are incubated at a temperature of from 16 to 22.degree. C.

6. A method for producing a catalytic subunit derivative of PP2A as
claimed in claim 5, wherein the catalytic subunit of PP2A is a catalytic
α-subunit or catalytic β-subunit.

7. A method for producing a catalytic subunit derivative of PP2A as
claimed in claim 5 or 6, wherein the tag is a polyhistidine tag.

8. A method for producing a catalytic subunit derivative of PP2A as
claimed in any of claims 5 to 7, wherein the substance capable of binding
to the tag is Ni.

Description:

TECHNICAL FIELD

[0001]The present invention relates to a method for producing heterodimer
derivatives of protein phosphatase 2A (hereinafter abbreviated to as
"PP2A"). More particularly, it relates to a method for production of a
large quantity of heterodimer derivatives of PP2A consisting of the
catalytic subunit and A subunit of PP2A, each carrying a tag, from which
derivatives a highly pure PP2A can be prepared.

BACKGROUND ART

[0002]PP2A is one of the most basic enzymes among hydrolases by which the
phosphate groups attached to serine/threonine residues in a protein are
hydrolyzed, and is formed as a complex of trimer in which a catalytic
subunit termed PP2Ac is linked to an A subunit and a B subunit. Among
these subunits, the catalytic subunit and the A subunit include two forms
of α-isoform and β-isoform.

[0003]The PP2A which plays an important role in signal transduction in
vivo is in big demand as a biochemical reagent and has come onto the
market at a high price. In addition, in recent years, there is a need as
a constitutive reagent for a convenient kit for measurement of toxic
components, okadaic acids, accumulated in marine Bivalvia, or blue-green
algal toxins (microcystins, nodularins), which are subjects to regulation
as poisonous ingredients in lakes and marshes.

[0004]At present, as for PP2A used for the above-mentioned objects, no one
but a heterodimer of the A subunit and the catalytic subunit has been
known, which is referred to as PP2A and prepared from the human blood
corpuscles.

[0005]The PP2A purified from the human blood corpuscles, however, is very
expensive because of complicated purification process and difficulty of
large-scale purification since it is separated from animal tissues. In
addition, there was a problem in using the resulting PP2A as a
biochemical reagent, since it is per se not likely to be sufficient in
purity. Further, there was another problem that a large quantity of pure
PP2A was unable to be produced at low cost or with easiness for the basic
research, making it difficult to achieve research necessary for
elucidation of a diversity of life process involving PP2A.

[0006]The present inventors previously studied a method for producing PP2A
and found that in producing the intended catalytic subunit and A subunit
of PP2A by a genetic engineering procedure using a baculovirus,
attachment of a tag to the respective subunits allows easy separation
from other proteins to obtain the subunits in a highly pure state. The
inventors further found that production of the catalytic subunit and A
subunit of PP2A carrying respectively different tags in the same cultured
cells obtained a heterodimer of PP2A consisting of the catalytic subunit
and A subunit respectively carrying different tags, and that use of these
two tags possessed by these subunits allowed easy purification of the
intended heterodimer only. A patent application was filed based on these
findings.

[0007]When a baculoviral expression system was used, however, there was
another problem that the rate of PP2A expressed as an insoluble protein
was higher than that as a soluble protein, thereby decreasing the rate of
PP2A to be purified, though the rate of expression was high.

DISCLOSURE OF INVENTION

Problem to be Solved by the Invention

[0008]Thus, an object of the present invention is to provide a means for
solving the problem that the rate of PP2A to be purified is low when a
baculovirus as an expression system is used.

Means for Solving the Problems

[0009]In order to solve the above problems, the present inventors have
assiduously studied the culture condition of insect cells particularly
after infection with baculovirus, and found that the soluble PP2A to be
purified was markedly increased in quantities when the insect cells were
incubated at a temperature lower than that employed in a usual culture
condition. Thus, the invention was completed.

[0010]The invention provides a method for producing a heterodimer
derivative of protein phosphatase 2A (PP2A) which comprises the steps of
infecting insect cultured cells with a baculovirus in which a cDNA
encoding the catalytic subunit of PP2A carrying a first tag is integrated
together with another baculovirus in which a cDNA encoding the A subunit
of PP2A carrying a second tag is integrated, incubating the infected
cells, then disrupting the incubated cells to obtain a disrupted cell
suspension, and then purifying the disrupted cell suspension with a solid
phase carrying a substance capable of binding to the first tag and
another solid phase carrying a substance capable of binding to the second
tag, characterized in that the insect cells infected with the baculovirus
are incubated at a temperature of from 16 to 22° C.

[0011]The invention also provides a method for producing a catalytic
subunit derivative of PP2A which comprises the steps of infecting insect
cultured cells with a baculovirus in which a cDNA encoding the catalytic
subunit of PP2A carrying a tag is integrated, incubating the infected
cells, disrupting the incubated cells to obtain a disrupted cell
suspension, and then purifying the disrupted cell suspension with a solid
phase carrying a substance capable of binding to the tag, characterized
in that the insect cells infected with the baculovirus are incubated at a
temperature of from 16 to 22° C.

[0012]In addition, the invention provides a method for producing an A
subunit derivative of PP2A which comprises the steps of infecting insect
cultured cells with a baculovirus in which a cDNA encoding the A subunit
of PP2A carrying a tag is integrated, incubating the infected cells,
disrupting the incubated cells to obtain a disrupted cell suspension, and
then purifying the disrupted cell suspension with a solid phase carrying
a substance capable of binding to the tag, characterized in that the
insect cells infected with the baculovirus are incubated at a temperature
of from 16 to 22° C.

Effect of the Invention

[0013]According to the invention, PP2A heterodimer derivatives having the
PP2A activity can be produced conveniently in large quantities with high
purity not through complicated extraction and purification steps of the
conventional techniques.

[0014]Therefore, the PP2A heterodimer derivatives obtained in the
invention can widely be utilized as PP2A of which the demand as a
biochemical reagent is expected to increase. In addition, it is expected
to be utilized in development of a convenient kit for measurement of
okadaic acids and the like.

BEST MODE FOR CARRYING OUT THE INVENTION

[0015]The term "derivative(s)" as used herein means that a tag is mainly
attached to an original compound. For example, a PP2A catalytic subunit
carrying a polyhistidine tag, FLAG tag, GST tag, HA tag, or the like is
defined as a PP2A catalytic subunit derivative.

[0016]In carrying out the invention, it is necessary to first obtain a
tagged catalytic subunit of PP2A and a cDNA encoding an A subunit of
PP2A. The preferred origin of the cDNA includes, but not particularly
limited to, animals such as human, mouse, rat, and the like, and cDNA of
human origin is particularly preferred.

[0017]In the cDNA, a tagged catalytic subunit of PP2A can be obtained, for
example, by preparing a plasmid clone containing a cDNA encoding α-
or β-isoforms of the PP2A catalytic subunit (PP2Ac) and a primer
corresponding to the tag, and carrying out PCR using the plasmid clone as
template. A cDNA encoding the tagged A subunit of PP2A may be obtained in
the same way as above by PCR using a plasmid clone containing a cDNA
encoding the A subunit of PP2A and a primer corresponding to the tag. In
each of the above-mentioned subunits, it is preferred for the tag to be
attached to the respective N-terminals.

[0018]The resulting cDNAs as mentioned above, which encode a tagged PP2A
catalytic subunit or a tagged PP2A A subunit, are respectively integrated
into a baculovirus, which is then introduced together into insect
cultured cells; the cells are incubated, from which the intended PP2A
heterodimer derivative is obtained. In this invention, it is important to
carry out incubation of the insect cultured cells infected with
baculovirus at a lower temperature than usual incubation temperatures
(about 27° C.); thus, the amount of PP2A heterodimer derivative
expressed is markedly increased. Specifically, the cells are incubated at
around 16 to 22° C., and more preferably at around 18 to
20° C.

[0019]According to the invention, the PP2A heterodimer derivatives can be
produced by carrying out a process comprising the following steps:
[0020](1) Integrating individually cDNAs encoding a catalytic subunit and
an A subunit of PP2A into cloning vectors; [0021](2) Integrating
individually the cDNAs encoding the respective subunits in the above
respective cloning vectors into recombinant donor plasmids for use in a
baculovirus expression system; [0022](3) Integrating individually the
cDNAs encoding the respective subunits by site-specific transposition
from the above recombinant donor plasmids into a baculovirus shuttle
vector (Bacmid); [0023](4) Preparing individually baculovirus in which
the cDNA encoding each subunit is integrated, from the above baculovirus
shuttle vector; [0024](5) Transfecting the resulting respective
baculovirus together into insect cultured cells to express the PP2A
heterodimer derivative, and incubating the cells at a temperature lower
than the above-mentioned usual incubation temperature; and [0025](6)
Purifying the expressed PP2A heterodimer in two steps.

[0026]In the above-mentioned process, the steps (1) and (2) are depicted
in FIG. 1, and the steps (3) to (5) in FIG. 2.

[0027]As for the cloning vectors used in the above step (1), TOPO vector
and the like may be used. As for the donor plasmids used in the above
step (2), pDEST8 and the like may be used. Further, in carrying out the
steps (3) and (4), for example, a commercially available expression
system, such as Bac-to-Bac baculovirus, may be used. Further, as for the
host cells to be infected with a baculovirus containing a cDNA encoding
each subunit, cells of armyworm or silkworm are utilized, and
particularly Sf9 cell, Sf21 cell, and High Five cell are preferred.

[0028]The invention is characterized in that the insect cultured cells
infected with a baculovirus are incubated at a lower temperature than the
usual incubation temperature (27° C.) in the above-mentioned step
(5), that is, at a temperature of around 16 to 22° C., preferably
at around 18 to 20° C. At such a temperature, the intended PP2A
heterodimer derivative can be produced in much higher yield, though it is
necessary to somewhat prolong the incubation period compared with the
case conducted at a usual incubation temperature.

[0029]Thus, the PP2A heterodimer derivative produced in the incubated
cells can be isolated from the disrupted cell suspension obtained from
the incubated cells by disruption. In carrying out disruption of the
incubated cells, such a procedure as ultrasonication or homogenization
may be employed. Thus resulting disrupted cell suspension is subjected to
separation from other contaminants in the step (6) using the tag attached
to each subunit. Specifically, the catalytic subunit and A subunit of
PP2A carry respectively distinct tags; thus, the suspension is
sequentially brought into contact with the respective solid phases
carrying the substances capable of binding to such tags, to obtain the
PP2A heterodimer derivative only.

[0030]In separation with these two tags, for example, when the tag
attached to the PP2A catalytic subunit is polyhistidine tag and the tag
attached to the A subunit is FLAG tag, the cells are disrupted by
centrifugation of the suspension, and then the resulting disrupted cell
suspension is brought into contact with a solid phase carrying a
substance capable of binding to the polyhistidine tag, such as
Ni-carrying solid phase. As for the solid phase, for example, agarose and
the like are utilized; the incubated suspension may be contacted with
solid phases in either batchwise or passing-through way.

[0031]Subsequently, the solid phase with which the disrupted cell
suspension has been contacted is washed with a suitable solvent, and then
treated with a processing solvent capable of cleaving the linkage with
the polyhistidine tag to obtain an eluate. When a target for binding to
polyhistidine is Ni, for example, 100-250 mM of imidazole and the like
are employed as the processing solvent.

[0032]Further, the above eluate is brought into contact with a solid phase
carrying a substance binding to FLAG tag. The substance binding to the
FLAG tag includes anti-FLAG M2 monoclonal antibody and the like; the
solid phase employed and the way of contact may be the same as mentioned
above.

[0033]Thereafter, the solid phase which has been contacted with the eluate
is washed well with a suitable solvent, and then treated with a
processing solvent capable of cleaving the linkage with the FLAG tag to
obtain an eluate. As the processing solvent, 0.2 mg/mL of FLAG peptide is
employed, for example.

[0034]On the other hand, when the tag attached to the catalytic subunit of
PP2A is HA tag and the tag attached to the A subunit is GST tag, the
disrupted cell suspension obtained in the same manner as above is brought
into contact with a substance capable of binding to HA tag, for example,
solid phase carrying an anti-HA antibody. As for the solid phase, for
example, agarose and the like are utilized; and the contact of the solid
phase with the incubated suspension may be achieved in either batchwise
or passing-through way.

[0035]Thereafter, the solid phase which has been contacted with the
disrupted cell suspension is washed with a suitable solvent, and then
treated with a processing solvent capable of cleaving the linkage with
the HA tag to obtain an eluate. As the processing solvent, when a target
for binding to the HA tag is an anti-HA antibody, 1 mg/mL of HA peptide
and the like is employed.

[0036]Further, the above eluate is brought into contact with a solid phase
carrying a substance binding to GST tag. The substance binding to the GST
tag includes glutathione and the like; the solid phase employed and the
way of contact may be the same as mentioned above.

[0037]Thereafter, the solid phase which has been contacted with the eluate
is washed well with a suitable solvent, and then treated with a
processing solvent capable of cleaving the linkage with the GST tag to
obtain an eluate. As the processing reagent, 5-10 mM of reduced
glutathione is employed, for example.

[0038]Thus resulting eluate only contains a PP2A heterodimer derivative as
an organic compound, and it can be utilized as a reagent and the like on
its own or after lyophilization. That is, since the PP2A heterodimer
derivative is purified by a two-step process, when a polyhistidine tag
and a FLAG tag are used as tags, for example, only the substances
carrying both tags are left in the product. The PP2A heterodimer
derivative is the only substance satisfying such a requirement in the
method of the invention.

[0039]Thus, a PP2A heterodimer derivative having very high purity can be
obtained by the above process.

[0040]In this connection, the PP2A catalytic subunit derivative can be
produced by infecting the cells of an insect independently with a
baculovirus in which a cDNA encoding a tagged catalytic subunit is
integrated in the step (5), and then carrying out the purification in the
step (6) using the tag attached to the catalytic subunit at one-stage;
but otherwise the above-mentioned steps (1) to (6) may be carried out
independently.

EXAMPLES

[0041]The invention is explained in detail by the following examples which
are not intended to limit the invention. In addition, since it is
apparent that an embodiment of the method as described in Examples can be
modified, such modification should not be interpreted to depart from the
scope of the invention, and all of modifications considered to be obvious
fall within the scope of Claims of the invention.

[0067]In the same manner as in Example 1, the DNA fragments amplified by
PCR were sub-cloned into the pENTR/SD/D-TOPO vectors to obtain the
recombinant donor plasmids, the recombinant baculovirus shuttle vectors
(Bacmid) were prepared, and the recombinant baculoviruses were produced
utilizing insect cultured cells, thereby obtaining 1.5 mL of culture
supernatant containing the recombinant baculoviruses.

Example 3

[0068]The recombinant baculoviruses in which cDNAs encoding the PP2A
catalytic subunits were integrated were infected to 200 mL of High Five
cells (2×106 cells/mL; 1 L spinner flask) and incubated at
19° C. for 4 days. After incubation, the cells were recovered,
from which recombinant proteins were purified according to the following
way.

[0069]The recovered cells re-suspended in PBS were centrifuged to collect
the cells and remove the supernatant, and the cells thus collected were
re-suspended in 20 mL of Buffer A (20 mM Tris-HCl, 2 mM EGTA, 0.5 mM
benzamidine, 0.5 mM DTT, 10% glycerol). The cells in the suspension were
disrupted by ultrasonication, and centrifuged with a small-size cooled
centrifuge (15,000 rpm, 20 min., 4° C.) to recover the
supernatant.

[0070]The resulting supernatant (5 μL) was applied to SDS-PAGE, and the
expression of PP2A catalytic subunit was confirmed by Western blotting.
The supernatant containing the PP2A catalytic subunit expressed as a
soluble protein was mixed and quickly agitated with 5 parts by volume of
ethanol, and centrifuged (4200×g, 15 min).

[0071]After centrifugation, the supernatant was removed, and the
precipitate was suspended in 20 mL of Buffer A. This suspension was
centrifuged (10,000×g, 20 min., 4° C.) to recover the
supernatant, 800 μL of Ni-NTA agarose (QIAGEN) equilibrated with
Buffer A was added to the supernatant thus obtained, and the mixture was
stirred moderately at 4° C. for 1 hour.

[0072]After a lapse of 1 hour, Ni-NTA agarose was recovered and washed
with Buffer A, and then 2.4 mL of eluate (Buffer A containing 250 mM
imidazole) was added thereto, and the mixture was stirred moderately at
4° C. for 30 minutes. After a lapse of 30 minutes, the eluate was
carefully recovered so that Ni-NTA agarose is not admixed.

[0073]By SDS-PAGE (0.2 μg of protein; 10% SDS-gel), the purified
recombinant protein in the recovered eluate was confirmed. The activity
of the purified protein was determined according to a method using
p-nitrophenylphosphate as substrate (Takai et al. 1995, Biochem J.
306:657-665).

[0074]The result of SDS-PAGE is shown in FIG. 3, and the activity to
p-nitrophenylphosphate is shown in FIG. 4, respectively. In both figures,
the term "27° C." indicates the tagged catalytic unit of PP2A
(comparative example), which was prepared in the same manner as in
Example 3 except that the cells infected with the recombinant baculovirus
was incubated at 27° C. for 3 days. In FIG. 3, the both end side
lanes indicate a marker, the 2nd lane from left indicates the tagged
catalytic unit of PP2A prepared by incubation at 27° C., and the
3rd lane from left indicates the tagged catalytic unit of PP2A prepared
by incubation at 19° C.

[0075]FIG. 3 shows that the tagged catalytic unit of PP2A prepared by
incubation at 19° C. can be obtained with lower production of
substances other than the intended compound. The result of FIG. 4 shows
that change of the incubation temperature from 27° C. to
19° C. for the recombinant baculovirus-infected cells enhanced 6
times or more the activity of the tagged catalytic unit of PP2A. In this
connection, the total yield of proteins was 240 μg/4×108
High Five cells when incubation was conducted at 19° C., while the
total yield of proteins was 88 μg/4×108 High Five cells
when incubation was conducted at 27° C.

Example 4

[0076](1) For the recombinant baculoviruses prepared in Example 2, the
amplification and infection with the virus was conducted, the infected
cells were recovered, and the recombinant protein was purified in the
same manner as in Example 3 except that 2×107 cells/mL of
cells were used. The recovered cells were washed with PBS in the same
manner as in Example 3, homogenized, and then centrifuged to obtain a
supernatant. The resulting supernatant was treated with 40 μL of
anti-FLAG M2 affinity gel (SIGMA) equilibrated with Buffer A, and stirred
moderately at 4° C. for 1 hour. After a lapse of 1 hour, the
anti-FLAG M2 affinity gel was recovered, washed with Buffer A, added 120
μL of eluate (Buffer A containing 0.2 mg/mL of FLAG peptide), and
stirred moderately at 4° C. for 30 minutes. After a lapse of 30
minutes, the eluate was carefully recovered so that the anti-FLAG M2
affinity gel is not admixed. By SDS-PAGE, the purified recombinant
protein in the recovered eluate was confirmed.

[0077](2) After confirmation of purification of the catalytic subunit of
human PP2A carrying a polyhistidine tag prepared in Example 3 and the A
subunit of human PP2A carrying a FLAG tag prepared in Example 4 (1), High
Five cells (2×107) were co-infected with the viruses used in
Examples 3 and 4, incubated at 19° C. for 4 days, recovered, and
used for purification of a heterodimer (His-PP2Acα/FLAG-PR65α
and His-PP2Acβ/FLAG-PR65β) recombinant proteins. Detail of
purification method is as follows.

[0078]First, the cells recovered in the same manner as in Example 3 were
washed with PBS, homogenized, and then centrifuged to obtain a
supernatant. The resulting supernatant was treated with 40 μL of
Ni-NTA agarose (QIAGEN) equilibrated with Buffer A, and the mixture was
stirred moderately at 4° C. for 1 hour. After a lapse of 1 hour,
Ni-NTA agarose was recovered and washed with Buffer A, 120 μL of
eluate (Buffer A containing 250 mM imidazole) was added thereto, and the
mixture was stirred moderately at 4° C. for 30 minutes. After a
lapse of 30 minutes, the eluate was carefully recovered so that Ni-NTA
agarose is not admixed. Through elution procedure repeated twice, the
first eluate (120 μL) and the second eluate (120 μL) were combined
to obtain 240 μL of eluate in total.

[0079]40 μL of anti-FLAG M2 affinity gel (SIGMA) equilibrated with
Buffer A was added to the eluate, and the mixture was stirred moderately
at 4° C. for 1 hour. After a lapse of 1 hour, the anti-FLAG M2
affinity gel was recovered and washed with Buffer A, 120 μL of an
eluate (Buffer A containing 0.2 mg/mL of FLAG peptide) was added thereto,
and the mixture was stirred moderately at 4° C. for 30 minutes.
After a lapse of 30 minutes, the eluate is recovered carefully so that
the anti-FLAG M2 affinity gel is not admixed. By SDS-PAGE, the purified
recombinant protein in the recovered eluate was confirmed. The activity
of the purified protein was determined according to a method using
p-nitrophenylphosphate as substrate (Takai et al. 1995, Biochem J.
306:657-665).

[0080]The tagged heterodimer (His-PP2Ac/FLAG-PR65) of the A subunit and
catalytic subunit of PP2A purified in Example 4 were analyzed by
SDS-PAGE. FIG. 5 shows the result. In the figure, the lane 1 indicates
the tagged PP2A heterodimer prepared by incubation at 19° C., and
the lane 2 indicates the PP2A heterodimer prepared by incubation at
27° C. FIG. 6 shows the results of investigation for the activity
of the heterodimers to p-nitrophenylphosphate. These results show that
according to the invention the tagged PP2A heterodimer can be prepared in
a pure form in larger quantities than in the incubation conducted at a
usual temperature of 27° C.

INDUSTRIAL APPLICABILITY

[0081]According to the method of the invention, it became possible to
produce the tagged catalytic subunit of PP2A and the tagged heterodimer
of PP2A A subunit and catalytic subunit in large quantities. Moreover,
these products have very high purity, and can advantageously be used for
the purpose of investigation of the inhibitory effect of okadaic acids,
microcystin or nodularin on the PP2A activity. Further, the above
heterodimers are able to bind to the B subunit of PP2A in an extract of
animal cells, and expected to widely be applied in the field of basic
research using PP2A.